1. Field of the Invention
The present invention relates to methods for enhancing fertility by reducing the activities and/or levels of circulating glycoprotein hormones having lutropin (LH) activity. The molecules of the invention are antibodies or other binding agents that reduce the biological activities of LH. The present invention also relates to novel methods for devising and/or selecting antibodies to specific portions of proteins including LH and human chorionic gonadotropin (hCG) to permit their biological activities to be reduced to desired degrees. The present invention further relates to novel glycoprotein hormone agonists and antagonists that reduce the activities of hormones with LH activities and/or increase the activities of hormones with follitropin activity.
2. Description of the Background
The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference and, for convenience, are numerically referenced in the following text and respectively grouped in the appended bibliography.
The glycoprotein hormone family (1) consists of three α,β heterodimeric glycoproteins found in the anterior pituitary gland where they are made. The glycoprotein hormones are luteinizing hormone (also known as lutropin or LH), follicle stimulating hormone (follitropin or FSH), and thyroid stimulating hormone (also known as thyrotropin or TSH). The hormones from humans are known as hLH, hFSH, and hTSH, respectively. In some species, a glycoprotein hormone structurally similar to LH called chorionic gonadotropin or CG, is made by the placenta and released into the circulation. In humans, this glycoprotein hormone is termed hCG. In primates, significant quantities of all the hormones are also found as excretion products in urine. After menopause, when the secretion of LH and FSH from the anterior pituitary is greatly increased, significant quantities of LH and FSH are found in the urine. Gonadotropin extracts of urine from menopausal women are termed human menopausal gonadotropins (hMG). Unlike hCG, which interacts like LH with LH receptors but only weakly with FSH receptors, hMG interacts with both LH and FSH receptors. The dual activity of hMG is due to the presence of hLH, hFSH, and their metabolites in the urinary extract. Urines from pregnant and menopausal women are major sources of gonadotropin activities and have important commercial uses.
Gonadotropins such as FSH, LH, and, in some species, CG play a major role in the reproductive process (1–6), while the structurally related hormone, TSH, is important for thyroid function (1). Both LH and FSH are essential for puberty and normal reproductive function. Lack of sufficient FSH, LH, or hCG at appropriate times results in infertility or termination of pregnancy. Excessive amounts of these hormones can result in premature puberty or hyperstimulation of the gonads. In the male, FSH is essential for the onset and maintenance of spermatogenesis (7,8). Immunoneutralization of FSH leads to a diminution in spermatogenesis and a loss in fertility. In the female, FSH is essential for follicular development leading to the production of the female gamete at ovulation. Polycystic ovarian disease is a common cause of infertility in women and is a condition characterized by incomplete follicular development. Fertility can usually be restored by administration of FSH or hMG. Fertility can also often be restored by treatments with antiestrogens, compounds that inhibit the negative feedback effect of estrogens on FSH secretion thereby allowing FSH levels to rise. In males, LH is required for puberty and, in its absence, there is a failure to acquire the sexual attributes and fertility of an adult. LH is primarily responsible for the synthesis of androgens in the testis. These steroids have a beneficial influence on spermatogenesis and abnormally high levels of androgens can maintain spermatogenesis once it has been initiated (9). In females, LH is essential for ovulation and formation of the corpus luteum. LH also has a synergistic influence with FSH on follicle development (4) and is well-known to promote the synthesis of follicular androgens. These androgens serve as precursors for FSH-stimulated estrogen formation. LH may also augment the effect of FSH on granulosa cells, particularly in the later stages of follicle maturation when the granulosa cells have acquired LH receptors. hCG made by the trophoblast is important for maintenance of progesterone secretion from the corpus luteum during early human pregnancy. The clinical activities of these hormones and their uses are reviewed extensively in several standard textbooks including that by Yen and Jaffe (2).
The differences in the effects of FSH and LH and the complex endocrine interactions between the two hormones cause them to have synergistic actions on follicular development and estradiol synthesis (4). For example, normal ovarian estrogen production is due to the effect of LH on androgen formation and the influence of FSH on the conversion of androgens to estradiol. In turn, estradiol can suppress FSH secretion from the pituitary gland. During the normal menstrual cycle, FSH levels decline as the follicle enlarges and secretes increasing amounts of estradiol. When estradiol levels reach a sufficient amount during the follicular phase, they can trigger an increase in LH secretion from the pituitary gland that causes ovulation. The ratio of LH/FSH activity as well as the absolute hormone levels in blood are important for reproductive functions such as follicle maturation and ovulation of the proper number of oocytes during the menstrual and estrus cycles.
While the secretion of both LH and FSH can be inhibited by steroid hormones, the secretion of FSH is usually more sensitive than that of LH to negative feedback regulation by estrogens. Indeed, in many species, high levels of estrogens can increase the secretion of LH, particularly if progesterone levels are low. Administration of anti-estrogens, compounds that disrupt the normal negative feedback regulation of estradiol on FSH secretion, often leads to increased FSH release and increased gamete production. Clinically, anti-estrogens are widely used to increase the probability of ovulation in women having polycystic ovarian disease. Unfortunately, since the negative effects of estradiol on FSH secretion are partly responsible for controlling the number of follicles that develop to the point of ovulation, disruption of the normal estrogen-FSH negative feedback loop can result in inappropriate numbers of ova being shed. A mechanism that results in increased FSH secretion without eliminating the negative feedback control of FSH secretion would have a valuable use in increasing fertility.
Purified FSH is capable of stimulating follicle development in women, particularly when some endogenous LH is also present. The ratio of FSH/LH is highest at the time of the menstrual cycle when follicular development is initiated. However, both hormones are essential for fertility. Immunoneutralization of LH leads to infertility in males and females. (10–12). Likewise immunoneutralization of CG, a hormone which acts via LH receptors was shown to block fertility in primates (13–16). Antibodies to LH have not been shown previously to stimulate fertility.
Monoclonal antibodies to hCG (termed hCG-mAb) have been shown to inhibit the binding of hCG to its receptor in vitro (17). Depending on the location of their epitopes, hCG-mAbs have differing abilities to inhibit binding of hCG to LH receptors. B105 and B110 are examples of monoclonal antibodies that recognize epitopes on hCG and LH that remain exposed when the hormones bind to LH receptors (17). Complexes of the hormones with these monoclonal antibodies bind to LH receptors, albeit with lower affinity than the free hormones. Consequently, these antibodies inhibit binding of the hormones to LH receptors. However, the maximal degree of inhibition observed in the presence of excess antibody is less than 100% and lower than that of antibodies which form complexes with the hormones that do not bind to LH receptors. In the presence of sufficient B105 or B110, the amount of hormone needed to induce a biological response is increased. Thus, even a massive excess of either antibody sufficient to bind virtually all the free hCG or LH in the assay is incapable of preventing a response to either hormone when the concentrations of the hormone-antibody complexes exceed a threshold level.
As discussed earlier, immunoneutralization of LH was shown several years ago to prevent fertility. This phenomena occurs because the antisera that were used in these studies neutralized the biological activity of LH. However, when appropriate antisera or antibodies like B105 or B110 are used, the biological activity of LH is not eliminated. Rather it is reduced by a predetermined amount. When this happens, androgen synthesis is reduced. Since androgens are precursors of estrogens, estrogen synthesis is also reduced. The decline in estradiol has a larger impact on FSH secretion than on LH secretion. The secretion of FSH will be enhanced and this will lead to an increased ratio of FSH/LH and enhanced follicular development. In females, this ratio of FSH/LH will lead to increased follicle development. In males, this ratio of FSH/LH will lead to increased Sertoli cell function and increased spermatogensis.
An approach to increasing fertility that is based on reducing LH levels has not been used previously. In part, this is due to the many reports that antibodies to LH inhibit fertility and because methods for making and selecting antibodies that reduce but do not neutralize LH activity were previously unknown. Thus, one would not expect that this approach to fertility would be successful. As will be discussed later, this approach to increasing fertility has several advantages relative to current techniques, principly in women who make and release LH and FSH from their pituitary glands. Since reducing LH levels does not disrupt the normal endocrine feedback relationships between estradiol and FSH on pituitary function, it has a much less likely chance to induce ovarian hyperstimulation than existing techniques. This means that there will be less need for expensive and demanding patient monitoring. In addition, only one or at most a few treatments will be required to induce fertility.
Another novel method for increasing fertility is to employ an LH antagonist during the follicular phase of the menstrual cycle. For several years it is known that the oligosaccharide chains on the glycoprotein hormones are essential for their abilities to elicit signal transduction (1). Glycoprotein hormones lacking carbohydrate residues have impaired abilities to elicit a biological response. These analogs can be used to block binding of LH to its receptors. This will reduce the activity of circulating LH and thereby improve fertility. Deglycosylated gonadotropins have been found to have short biological half-lives and were found not to be useful for their original intended use, namely to inhibit fertility by reducing luteal progesterone synthesis and causing abortion. By moving the carbohydrate residues to alternate portions of the hormone by removing glycosylation signals (i.e., the amino acid sequences Asparagine-X-Threonine or Asparagine-X-Serine, where X is any amino acid except Proline) from one site and by creating glycosylation signals at alternate sites of the α- and β-subunits, it is possible to design analogs with reduced agonist activity that have sufficiently long half-lives to be useful. In addition, by preparing single chain gonadotropins in which the α- and β-subunits are covalently linked, it is possible to increase the stability of the hormones in circulation. This is because the receptor binding activities and the plasma half-lives of the heterodimeric gonadotropins are greater than either of the subunits. Covalent linkage prevents the dissociation of the two subunits in circulation.
While stimulation of fertility is important to restore fertility to infertile couples, inhibition of fertility is often desirable as a method of family planning. In addition, inhibition of fertility would be useful in the commercial production of livestock since it would eliminate the need for castration or it would prevent the development of heat in cattle held in the feedlot. Inhibition of fertility in other animals including dogs and cats would also be desirable as a replacement for spaying or castrating them. Inhibition of fertility in horses would also be preferable to gelding, particularly if it can be reversed. As noted above, fertility can be inhibited by administration of neutralizing antibodies to LH or FSH. It can also be inhibited by using a vaccine to induce the formation of these antibodies. Due to the action of hCG in maintaining pregnancy, treatments that lead to diminished hCG secretion or activity would also be expected to cause infertility. In women, it would be more desirable to inhibit fertility by inhibiting hCG rather than hLH or hFSH. This is because treatments that neutralized hLH or hFSH would cause cessation of ovarian function and hasten the onset of problems associated with menopause. In cattle and other domestic animals, it would be more important to inhibit LH to prevent puberty or to disrupt heat. As noted earlier, appropriate antibodies to chorionic gonadotropin are able to inhibit fertility in primates and women and the development of antibodies to hCG has been recognized to be an important potential method of contraception for many years (18). Since hCG is produced by a large number of human cancers and since antibodies to hCG can disrupt these tumors, immunization would also have a beneficial impact on cancer therapy or prevention (19).
Several attempts have been made to devise such an hCG-based contraceptive vaccine taking into account the differences between hCG and the other glycoprotein hormones (14,18). Unfortunately, development of the vaccine has been hampered by the structural homologies between all the glycoprotein hormones. The preferred immunogen must be highly antigenic yet not induce; antibodies that crossreact with the other glycoproteins such as human FSH, LH, or TSH. Based on the knowledge of glycoprotein hormone activities outlined above, a vaccine that induced antibodies that interacted with LH, FSH, or TSH would also cause infertility and/or inhibition of thyroid function. Unfortunately, neutralization of LH or FSH would also result in cessation of normal menstrual cycles and the loss of estrogen production that is associated with fertility in women. Termination of ovarian function would be likely to result in premature development of osteoporosis and other problems associated with menopause. Inhibition of thyroid function would lead to hypothyroidism. Similarities in the structures of hCG and hLH have made it particularly difficult to design an appropriate immunogen that does not generate crossreacting antibodies. Most efforts have been devoted to making antibodies against the unique C-terminus of the hCG β-subunit since this portion of the molecule is not found in hLH (1). However, this region is not very antigenic. Efforts to devise immunogens have also employed peptides obtained from the β-subunit (14), conjugates of the β-subunit with other proteins (20), or heterodimers containing hCG β-subunits conjugates, and ovine α-subunits (18). Unfortunately, most of these immunogens are not very effective and a better immunogen is needed to make this method practical.
The difficulty of devising a vaccine based on hCG can be appreciated by an understanding of the structures of the glycoprotein hormones. All of the glycoprotein hormones contain a common α-subunit. While the conformation of parts of the α-subunit differ in all the hormones and can be recognized by selected monoclonal antibodies (21), portions of the α-subunit have the same conformation in each glycoprotein hormone. Thus, many antibodies to the α-subunit recognize LH, FSH, hCG, and TSH. Since anti-α-subunit antibodies are often capable of blocking the activities of the hormones (22), an immunogen which induced a response to the α-subunit is likely to have unwanted side effects. Therefore, most strategies for devising a contraceptive vaccine are directed at the hormone specific β-subunit of hCG.
The β-subunit of hCG is most closely related to the β-subunit of hLH. Many antibodies directed against the intact hCG β-subunit will also combine with the LH β-subunit. While the β-subunits of the other hormones differ considerably from that of hCG, some of the residues in all the β-subunits are identical and there is the possibility, albeit small, that some anti-β-subunit antibodies will crossreact with these hormones as well. The carboxy terminal 31 amino acids of the hCG β-subunit (CTP) are unrelated to any of the residues in the other glycoprotein hormones. In theory, antibodies to this region cannot elicit any crossreaction with the other hormones. As expected, when this region is used as an immunogen, antibodies are developed that do not crossreact with any of the other glycoprotein hormones. Unfortunately, the antibodies that are produced to synthetic CTP peptides do not bind with high affinity to hCG. In part, this is due to the observation that this region of hCG contains four potential serine-linked glycosylation sites and is highly glycosylated. Furthermore, much of this region of hCG is not essential for interaction with LH receptors. Thus, the antibodies directed against the CTP of hCG bind to hCG receptor complexes and are primarily of the nonneutralizing type. Consequently, they do not inhibit hCG action similar to antibodies like B101 (22) that prevent hCG from binding to LH receptors.
Efforts have also been made to devise antibodies against other portions of the hCG β-subunit. One region that has been investigated extensively is that found between cysteine residues 38 and 57. This portion of the protein is known to form a large loop and studies have shown that this loop is capable of stimulating steroidogenesis (23,24). Thus, one would anticipate that antibodies against this loop would be of the neutralizing type. Indeed, B101, an antibody which has been shown to recognize residues within this loop (22,25,26) is capable of neutralizing hCG activity. The problem with using this loop structure is that the antibodies that are produced are often of low affinity. In addition, since hCG and hLH are similar in this region of the molecule (i.e., there are only three amino acids that differ), immunization with this loop is expected to cause the production of antibodies against hLH. Indeed, B101, an antibody that binds to this region of the molecule has an unacceptably high affinity for hLH.
Recent efforts at identifying the tertiary structure of the glycoprotein hormones have depended on characterizing the binding sites of panels of monoclonal antibodies (26). Antibodies have been identified that prevent the biological activity of hCG or that only partially neutralize its biological activity. As outlined in example 7 of the present specification, these and similar antibodies can be used to devise immunogens that have the potential to neutralize hCG but not hLH using the positive and a negative selection procedure outlined in examples 6 and 7 set out below. While the hormone has been crystallized and a crystal structure would be valuable in determining the types of immunogens that would give a high titer immune response to particular parts of the molecule, difficulties in solving the crystal structure have precluded this approach. Thus, at the present state of the knowledge of hCG structure, there is no good method that could be used to predict the type of immunogen that would be most effective.
Another useful method for increasing fertility is to increase the levels of FSH activity. One way of accomplishing this is to administer small doses of long-acting follitropins. These can be made by coupling molecules with follitropin activity to molecules with long plasma half-lives (i.e., immunoglobulins) or by preparing single-chain gonadotropin analogs having follitropin activity (Tables 1 and 2). Alone, or in combination with antibodies to LH and/or LH antagonists, these hormones facilitate follicle development in women with polycystic ovarian disease.